Studies On Host Proteins Related To Replication And Proliferation Of Japanese Encephalitis Virus Based On CRISPR/Cas9

The Japanese Encephalitis Virus(JEV)is a single-stranded positive-sense RNA virus with small genome structural and simple protein construction,which requires host factors to complete the life cycle.Our previous study found that host proteins ZNF251 and ?2M interacted with the NS1 protein and host proteins CRBN,PPP2 CB,FBLN-5 interacted with the NS1 protein.This study aims to the effect of those host proteins on the replication and proliferation of JEV.First,the 293 knockout cell lines were constructed successfully.The 293 knockout cell line were successfully constructed by CRISPR/Cas9 and correct identification was completed subsequently by sequencing and Western Blotting.Then,ZNF251 protein expression in 293 cells was inhibited by CRISPR/Cas9 and found that positively regulated JEV mRNA levels and progeny virus particles,but similar results were not found in experimental group of others.The RT real-time fluorescence quantification PCR and plaque form assay were executed after the JEV infected the knockout cell line.The JEV mRNA level detected on ZNF251 K.O cells was found to be approximately 2.4-fold higher,and the progeny virus titer was lg8.67 PFU/ml,comparing with negative control group of that lg7.80 PFU/ml.However,other knockout cell line transfected with the virus but show no significant difference on viral mRNA level and progeny virus titer with the control group.In conclusion,CRISPR/Cas9 could be successfully used in 293 cells for gene editing and JEV related research subsequently,found that ZNF251 is a host protein of 293 cells that affects the proliferation and replication of JEV,which has a certain inhibitory effect.